Alcohol (ethanol) is the most widely abused substance in the world. Unfortunately, medical treatments to reduce complications of alcohol abuse face challenges including lack of adherence, low efficacy, and serious side effects. Therefore, there is a need to discover new therapies acting at novel targets. This study will examine the effects of ethanol on Glycine receptors (GlyRs), one of the most sensitive targets for this drug. Previously, we made significant progress determining the role of basic residues in the intracellular loop (IL) of the ?1 subunit of GlyRs for ethanol. These residues are important for potentiation with low ethanol concentrations, without affecting ion channel physiology and sensitivity to other modulators. GlyRs are critical for control of excitability in the spinal cord and brain stem, regulating important functions such as pain transmission, respiratory rhythms and motor coordination. Studies in spinal and hypoglossal neurons expressing ?1 subunits showed that they are sensitive to relevant concentrations of ethanol, suggesting that reduction in muscle control and respiratory depression might result from their modulation. Furthermore, GlyRs in nucleus accumbens (nAc) and the VTA were recently implicated in ethanol intake and seeking behavior. However, their pharmacological relevance and molecular composition are still unclear. There are three important questions still not answered regarding the effects of ethanol on GlyRs: 1) What is the role of the ? subunit on the effect of ethanol? 2) How sensitive are supra-spinal GlyRs to ethanol? and 3) What is the molecular composition of supra spinal GlyRs? These questions arise because most studies use ?1 homomeric GlyRs since the ?1 subunit alone forms a GlyR complex, which has the binding site for the agonist and the ionic channel. In addition, although GlyRs are expressed in upper brain regions (above brain stem), there is no compelling data on the sensitivity of this complex to ethanol or on its molecular composition. Our data indicate that the ? subunit is a critical ethanol regulatory component. We found that co expression of ?2? remarkably increased the ethanol sensitivity (>1 mM). This conformation showed a significant potentiation from 5-10 mM ethanol, which is a concentration near the legal limit of alcohol for drivers. Furthermore, recording of glycine activated currents in GFP-D1R MSN in the nAc showed that they are potentiated by ethanol. We plan to focus our research on the following three specific aims: SA1. Characterize the modulatory effect of the ? subunit on ethanol actions in GlyRs. SA2. Characterize the effects of low ethanol concentrations on a critical mesolimbic region (i.e. nAc). SA3. Characterize ethanol behaviors on genetically engineered mice with loss or gain of GlyR function. We expect that by integrating state of the art research in cell biology, biochemistry and genetically-modified mice, we will characterize a new molecular target relevant for ethanol toxicity and abuse. Thus, the overall and specific significances of this study, with highly recognized USA scientists, are well within NIAAA interests.